DEV004598 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Specific reaction of guinea pig antiserum to bacterially expressed At-Fkh. SDS-PAGE of lysates of BL21 (DE3) cells transformed with pMAL-At-Fkh (lanes 1 and 2) or pMAL-cRI (New England Biolabs) (lanes 3 and 4) followed by Coomassie brilliant blue staining (CBB, top) or followed by western blotting with rabbit anti-maltose-binding protein antiserum (anti-MBP, middle) and guinea pig anti-At-Fkh antiserum (anti-At-Fkh, bottom). For western blotting, cell lysates diluted 1:50 were loaded on each lane. Expression of each transgene was induced by 0.3 mM IPTG (lanes 1 and 3) or not induced (lanes 2 and 4). For construction of pMAL-At-Fkh, a cDNA fragment encoding the C-terminal 182 amino acids of At-Fkh (aa 225-406), which was the same as used for the pET-At-Fkh fusion construct (see Materials and methods), was inserted between the KpnI and SalI sites of pMAL-cRI. In the CBB-stained gel, strong bands of the MBP-At-Fkh and MBP-β-gal fusion proteins are seen in lanes 1 and 3, respectively. Anti-At-Fkh antiserum reacted to the MBP-At-Fkh fusion protein but not to the MBP-β-gal fusion protein. Anti-MBP antiserum (New England Biolabs) was used at a dilution of 1:5000, and anti-At-Fkh antiserum at a dilution of 1:1000.

• Supplemental Figure S2 -

  Fig. S2. Time course of phenotype expression in caudal lobe after injection of At-Notch or At-Su(H) dsRNA. Each graph refers to one female injected with At-Notch (A) or At-Su(H) (B) dsRNA. Each vertical bar indicates the relative numbers of embryos exhibiting normal (blue), invagination (red) and non-specific (yellow) phenotypes in each egg sac. The number at the top of each bar indicates the total number of embryos examined. Not all egg sacs produced by the females are presented. Unfertilized eggs and embryos failing to form morphologically normal germ disc were excluded from the analysis. ‘Non-specific’ phenotypes, often observed even in embryos derived from untreated females, include shrinking, thickening and destruction of germ disc.

• Movie 1 -

  Movie 1. Time-lapse observation of live embryos derived from females injected with gfp (control, left) or At-Delta^EB (At-Delta, right) dsRNA. These embryos are the same as those shown in Fig. 4A,B. Time (hour) is shown at the left side of the top in each panel. Developmental stages are indicated at the left side of the bottom. From early stage 6, a large invagination develops at the center of the germ disc in the right embryo but not in the left embryo. The progression of shaping is relatively slow in the right embryo, in which limb bud formation is largely delayed.